We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four ␣-helices and Val 362 was found to be located in the middle of the 3rd helix. Replacement of Val 362 with amino acid residues that disrupt the ␣-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the ␣-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val 362 is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.